Abstract: HIV-associated neurocognitive disorder (HAND) persists in spite of effective control of HIV replication by modern antiretroviral therapies. Adjunctive therapies treating HAND in virally suppressed patients are needed. Studies over the previous funding period suggest that cognitive impairment in HAND is correlated to decreased dendritic spine density in prefrontal cortex (PFC) neurons, which is influenced by several factors such as HIV proteins, chronic inflammation, and opioid use. Importantly, this process may be reversible. Our previous work revealed that activation of the CXCL12/CXCR4 chemokine axis increases dendritic spine density in medial PFC neurons likely leading to cognitive improvement. On the other hand, studies in multiple species - including humans - demonstrate that opioids compromise this pathway, representing a potential mechanism of accelerated HAND. With the long-term goal of introducing targeted therapeutics for HAND, this study will dissect the mechanisms whereby the CXCL12/CXCR4 axis regulates spatiotemporal expression of discrete dendritic spines in PFC neurons. Additionally, the proposed studies will determine the relationship of the spine changes with cognitive function and how opioids or HIV impact these structural and functional outcomes. Experiments will focus on the Rac1/PAK pathway, a main regulator of spine stabilization coupled to the CXCR4 receptor. We hypothesize that CXCL12/CXCR4 signaling enhances working memory and cognitive flexibility by stabilizing normally transient thin spines. Conversely, impairment of CXCR4/Rac1/PAK contributes to synaptic and cognitive deficiencies associated with HIV infection and opioid exposure. Studies in Aim 1 will dissect the signaling pathways downstream of CXCR4 that regulate dendritic spines in cortical neurons, and characterize the effect of CXCL12 on spine dynamics. The proposed experiments will establish the importance of the Rac1/PAK pathway in CXCL12-induced spine regulation and cognitive performance. Aim 2 will examine the effects of morphine on spine morphology and function, in the presence and absence of CXCL12. Although CXCL12 may work to enhance stability of dendritic spines, the development of a transient spine to one that is integrated into the neuronal circuitry is dependent on additional factors, including neuronal activity and protein synthesis. Since morphine and other -opioid drugs affect neuronal activity in various ways, these compounds may have additional effects on spines that are unrelated to CXCR4 inhibition. Finally, Aim 3 will define alterations of synaptodendritic architecture and Rac1/PAK pathway activity after in vivo exposure to HIV proteins and morphine, using a small animal model of HAND. These studies will also determine whether CXCL12 treatment rescues synaptic deficits and cognitive performance in a PFC-mediated behavioral task highly relevant to HAND. Behavioral studies will be linked with downstream spine and protein/gene expression analysis in the same group of animals to establish the causal link between changes in cognitive ability and altered synaptic density or connectivity.